Oral dosage composition

ABSTRACT

The present invention is an oral dosage composition for prevention and treatment of hepatic inflammation such as NASH. The present invention is an oral dosage composition for prevention or treatment of hepatic inflammation, contains the radical scavenging active spirulina powder effective in preventing, ameliorating, and curing NASH or the like.

TECHNICAL FIELD

The present invention relates to oral dosage compositions for at leastone of prevention and treatment of hepatic inflammations such asnonalcoholic steatohepatitis (which is referred to as “NASH”).

BACKGROUND ART

Fatty liver can be accompanied by hepatic inflammation to cause livercirrhosis. Even non-alcohol drinkers can suffer from a symptom analogousto an alcoholic steatohepatitis. The symptom is referred to as anonalcoholic steatohepatitis (NASH), and has attracted attention as anew life-style related disease as well as high blood pressure, diabetes,hyperlipemia.

With increase in the numbers of obese people and patients suffering fromlife-style related diseases due to the shift to western-style diets suchas lipid-rich diets, and lack of physical activity, the numbers of fattyliver patient and NASH patient presumably increase. Hence, it isnecessary to develop new medicines for NASH treatment, suppressants forsuppressing NASH development, and/or functional foods for reducing riskof NASH occurrence and NASH development, and establish methods of curingNASH and preventing NASH development.

Human NASH is diagnosed by identification of biochemical andhistopathological characters of human blood. NASH pathological animalmodel can be prepared from rats with a partial pressure of oxygen in ratblood being kept at a low level, for evaluation of unknown functionalmaterials.

Non-patent reference 1 (Takayama F. et al: J. Pharmacological Sci., 100(1), pp. 164 (2006).) describes in detail a relationship between theNASH pathological animal model and an oxidative stress. In thisreference, the NASH pathological rat model promotes hydroxide radicalproduction at its liver mitochondria, suggesting that it is possible toprevent, alleviate, and cure NASH by promoting hydroxyl radicalscavenging activity at its liver mitochondria.

Generally, active oxygen species (ROS) is widely known as an origin ofdiseases, for example life-style related diseases such as cancer,cataract, nerve disease, kidney disease, allergy, and diabetes. Ingeneral, the ROS involving diseases are effectively suppressed byantioxidants.

Non-patent reference 1 shows an experiment using NASH pathological ratmodel prepared similarly to NASH pathological human model in which livermitochondria promotes active oxygen radical production, stronglysuggesting a relationship between oxidative stress and NASH.

As containing phycocyanin as an effective ingredient, the driedspirulina (e.g., Spirulina platensis) powder has been consumed as asupplement, and can be widely utilized as ingredients of cosmetic andmedical products or the like, as described in patent reference 1(Japanese unexamined patent application publication No. 2007-215507),patent reference 2 (Japanese unexamined patent application publicationNo. 2004-238519) and patent reference 3 (Japanese unexamined patentapplication publication No. 2004-256478).

The health functionality of dried spirulina can be attributed to itsactivities for scavenging ROS, especially lipid-peroxidate (seenon-patent reference 2; Vadiraja B. Bhat and K. M. Madyastha: Biochem.Biophys. Res. Commum., 275, pp. 20-25 (2000).), hydroxyl radical (seenon-patent reference 3; Pinero Estrada et al: II Farmaco., 56, pp.497-500 (2001).), peroxynitrite (see non-patent reference 4; Vadiraja B.Bhat and K. M. Madyastha: Biochem. Biophys. Res. Commum., 285, pp.262-266 (2001).), and so on.

The dried spirulina product is known as an ingredient effective inpreventing and curing various diseases. For example, the dried spirulinaproduct can bring various effects such as blood cholesterol suppressioneffect (non-patent reference 5; Toshimitsu Kato et al., Publication ofJapanese Society of Nutrition and Food Science 37(4), 323-332 (1984).),hyperlipidemia alleviation effect (non-patent reference 6; Kazuko Iwataet al., Publication of Japanese Society of Nutrition and Food Science 40(6), 463-467 (1987).), blood pressure regulation effect (non-patentreference 7; Kazuko Iwata et al., Kagawa Education Institute ofNutrition abstract, 21, 63-70 (1990).). The dried spirulina product canhave a UV absorption effect for being applied to skin care medicine (seepatent reference 2), as well as immune system enhancement effect andallergic inflammation suppression effect (see non-patent reference 8;Hayashi O. et al., J. Nutr. Sci. Vitaminol., 44, 841-851 (1998) andpatent reference 3).

The application of the dried spirulina products to foods and pigmentshave been proposed as described below, and partially achieved forpractical use. The dried spirulina product can be applied to foods (seepatent reference 1 and patent reference 4 (Japanese unexamined patentapplication publication No. 1995-289201). Phycocyanin extracted from thedried spirulina product can be available as a food pigment, as proposedin patent reference 5 (Japanese unexamined patent applicationpublication No. 2001-190244) and patent reference 6 (Japanese unexaminedpatent application publication No. 2006-230272).

Although the dried spirulina product has a high health functionality,radical scavenging active level of the dried spirulina product forutilizing the functionality has not been determined yet. Besides, theapplication of dried spirulina product has not been established yet, asto specific use of fermentation products for treatment of life-stylerelated diseases involving active oxygen species.

In view of this circumstance, it is necessary to meet increasing demandfor establishment of methods for applying the highly radical-scavengingactive dried spirulina products to treatment of life style-relateddiseases, especially hepatic inflammation such as NASH.

DISCLOSURE OF THE INVENTION

The present invention has been accomplished in view of the aboveproblems, and is intended to provide an oral dosage composition forprevention and treatment of hepatic inflammation such as NASH.

Inventors of the present invention have intensively studied to solve theabove problems, and accomplished the present invention for achieving (1)establishment of a highly radical-scavenging active dried spirulinaproduct and techniques for preparation of the same, (2) development of aspirulina-containing synthetic food containing the dried spirulinaproduct, and (3) elucidation of a relationship between NASH and theradical scavenging-activity of the dried spirulina product.

First, the oral dosage composition recited in claim 1 of the presentinvention is characterized in that it is utilized for at least one ofprevention and treatment of hepatic inflammation, and comprisesspirulina as an effective ingredient.

Secondly, the oral dosage composition recited in claim 2 of the presentinvention is characterized in that the hepatic inflammation is NASH.

Thirdly, the oral dosage composition recited in claim 3 as set forth inclaim 1 or 2 of the present invention is characterized in that itcomprises 0.1% or more by weight of dried spirulina powder.

Fourthly, the oral dosage composition recited in claim 4 as set forth inany one of claims 1 to 3 of the present invention is characterized inthat it has a hydroxyl radical scavenging activity with IC₅₀ valueranging from 3000 μg/ml to 100 μg/ml determined by an electron spinresonance spectroscopy—spin-trapping method.

The oral dosage composition recited In claim 1 of the present inventionenables it to prevent, alleviate and cure the hepatic inflammationmainly due to the radical scavenging activity of spirulina powder, whenorally ingested by patients.

The oral dosage composition recited in claim 2 of the present inventionenables it to prevent, alleviate and cure NASH mainly due to the radicalscavenging activity of spirulina powder, when orally ingested bypatients.

The oral dosage composition recited In claim 3 of the present inventionenables it to facilitate the prevention, alleviation and treatment ofthe hepatic inflammation such as NASH.

The oral dosage composition recited In claim 4 of the present inventionenables it to facilitate the prevention, alleviation, and treatment ofthe hepatic inflammation such as NASH mainly due to theradical-scavenging activity of spirulina powder.

BEST MODE FOR CARRYING OUT THE INVENTION

Explanations are given below as to best mode for carrying out theinvention.

Spirulina in the present invention is exemplified by Spirulinacorakiana, Spirulina crispum, Spirulina labyrinthiformis, Spirulinalaxa, Spirulina laxissima, Spirulina major, Spirulina maxima, Spirulinameneghiniana, Spirulina nordstedtii, Spirulina platensis, Spirulinaprinceps, Spirulina subsalsa, Spirulina subtilissima, Spirulinatenerrima, Spirulina weissii, Spirulina fusiformis, Spirulina jenneri,and the like. In preparation of dried spirulina powder, for example,incubated pure seeds of the above-listed spirulina are cleanly incubatedin a pool filled with alkali aqueous solution having a pH of 8 to 11 ina predetermined period, in order to grow spirulina algae. The obtainedspirulina algae is isolated, condensed, cleaned, filtered, and dried toprovide the dried spirulina powder. The dried Spirulina powder isprovided as a food meeting hygiene requirements, as well as havingradical scavenging activity.

In this oral dosage composition of the present invention, the driedspirulina can be utilized without further modification. The driedspirulina can be processed into particles, microparticles, or tabletsfor being easily ingested. The dried spirulina may be mixed withcoloring agents, flavors, seasoning agents, or other food additives.

The oral dosage composition in the present invention can be ingested asother foods. The present invention is not limited by particular maximumsand minimums of dosage amount, dosage method and dosage period, andother requirements. The oral dosage composition in the present inventioncan be taken in various ways such as short-time large-dose or long-timesmall-dose, and or doses combined with other health functional material.But, the present invention is not limited by the above ways. Forexample, the recommended human dose of this composition may be set in arange of 2 to 10 grams per day.

The oral dosage composition in the present invention can be preparedfrom the dried spirulina powder alone, or a mixture of the driedspirulina powder and other ingredients such as conventional foods. Whenadded into foods, the dried spirulina powder has a content of 0.1% ormore, preferably 1.0% or more, further preferably 5.0% or more by weightwith respect to the foods, for providing the oral dosage composition ofthe present invention as a preferable food effective in preventing andalleviating NASH. But, the maximum content of spirulina in food is notparticularly limited in the present invention. The oral dosagecomposition in the present invention need not be combined with othermaterials, but may be solely utilized to provide a food with 100% oraldosage composition content. The food for mixed with this oral dosagecomposition may be powder-like, liquid-like, paste-like, or the like.The content of this oral dosage composition in the food and the way toingest this oral dosage composition are not particularly limited in thepresent invention, and can be suitably determined depending on cost andquality requirements such as functionality, and requirements forprevention and treatment of NASH (e.g., species and extent of pathology,dose initiating-time and prescribing time period for ingesting, combinedmaterials, and others).

The oral dosage composition in the present invention can be employed asa composition with its hydroxyl radical-scavenging activity forprevention, alleviation and treatment of NASH pathology. Theradical-scavenging activity can be determined in terms of an IC₅₀ valueby an electron spin resonance—spin trapping method. When having the IC₅₀value in a range of 3000 μg/ml to 100 μg/ml, the oral dosage compositionenables to effectively achieve objects of the present invention. Whenhaving IC₅₀ value less than 100 μg/ml, this composition can notsufficiently provide the objects of the present invention. When havingIC₅₀ value more than 3000 μg/ml, this composition may bring biologicaladverse effects due to its excessive radical-scavenging activity. It isnoted that each of the above concentrations is the final concentrationin an ESR sample solution. The hydroxyl radical-scavenging activity ofthis oral dosage composition is evaluated relative to that of Trolox,The above hydroxyl radical-scavenging activity corresponds to IC₅₀ valuein a range of 0.14 to 4.14 μmol (Trolox equivalent/mg) with respect tothat of Trorox. The oral dosage composition of the present inventionpreferably has the radical-scavenging activity with IC₅₀ value less thanthat of Trolox determined by ESR measurement, for being safely utilizedas a radical-scavenging active composition for prevention, alleviationand treatment of NASH pathology. The oral dosage composition withhydroxyl-scavenging activity more potent than Trolox may bringunpredictable biological adverse effects, when excessively consumed.

The hydroxyl-scavenging activity of the oral dosage composition in thepresent invention can be evaluated by means of electron spinresonance—spin trapping method as follows. X-band ESR apparatus (RX-typeavailable from JEOL Ltd.) is employed as a radical detection apparatus,and equipped with a digital high-speed sweeping unit (available fromRadical Research Inc.) and an WIN-RAD system RDA-03W ESR data analyzer(available from Radical Research Inc.).

5,5-dimethyl-1-pyrroline-N-oxide (DM PO, available from LABOTEC Co.,Ltd.) or2-(5,5-Dimethyl-2-oxo-2-λ5-[1,3,2]dioxaphosphinan-2-yl)-2-methyl-3,4-dihydro-2H-pyrrole 1-oxide (CYPMPO available from Radical Research Inc.) isemployed as a spin trapping agent. The ESR spectrum were recorded withthe following spectrometer parameter settings: sweep range of magneticfield, 336.5±5 mT for DMPO-containing solution or 331.5±10 mT forCYPMPO-containing solution; field modulation, 0.079; time constant, 0.10sec; sweep time, one minute for DMPO-containing solution or four minutesfor CYPMPO-containing solution; output power, 8.0 mW. The evaluation isperformed in terms of a relative intensity of the ESR spectrum of DMPOor CYPMPO spin adduct trapping radicals.

In the present invention, the hydroxyl radical-scavenging activity ofthe sample (the oral dosage composition) is determined in terms of IC₅₀value (μg/ml, final concentration) representing a concentration of theoral dosage composition causing 50% reduction in ESR spectral intensitycompared to that of a control solution without containing thiscomposition. Besides, the hydroxyl radical-scavenging activity of Trolox((±)-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid availablefrom Aldrich Inc.) is also determined in terms of IC₅₀ value (μg/ml,final concentration) representing a concentration of Trolox causing 50%reduction in ESR spectral intensity compared to that of a controlsolution without containing Trolox. Then, the hydroxylradical-scavenging activity of the oral dosage composition is determinedin terms of IC₅₀ value with respect to that of Trolox having the sameactivity. Namely, Trolox is utilized a reference material, forevaluation of hydroxyl radical-scavenging activity of the sample (theoral dosage composition made of dried spirulina) in terms of a chemicalequivalent of Trolox giving the same activity as this sample. In thismeasurement, it is possible to obtain data with high objectivity andhigh quantitativity by minimizing errors dependent on ESR apparatus,measurement way, agent purity, and the like.

As described above, the present invention has been achieved to providethe oral dosage composition containing highly radical-scavenging activedried spirulina powder as an effective ingredient for effectiveprevention, alleviation and treatment of NASH when orally administeredto patients.

EXAMPLE

Hereafter, explanations are specifically given as to the presentinvention with reference to Example. The following Example is intendedonly to give examples of the present invention. The present invention isnot limited to the following Example in any sense.

<Measurement of Hydroxyl Radical Scavenging Activity for the Oral DosageComposition in the Present Invention (Example 1) and Trolox>

The oral dosage composition in the present invention was prepared asExample 1. The oral dosage composition in Example 1 contains 100%spirulina powder content.

The activity of scavenging free radical and reactive oxygen species wasmeasured by the electron spin resonance (ESR)—spin trapping method forthe oral dosage composition in Example 1 prepared in the above way.X-band ESR apparatus (RX-type available from JEOL Ltd.) was employed asa radical detection apparatus, and equipped with a digital high-speedsweeping unit (available from Radical Research Inc.) for measurementwith high sensitivity at high speed. Besides, the ESR apparatus wasconnected to an WIN-RAD system RDA-03W ESR data analyzer (available fromRadical Research Inc.). CYPMPO was employed as a spin trapping agent.

The ESR spectrum was recorded with the following spectrometer parametersettings: sweep range of magnetic field, 336.5±5 mT for DMPO-containingsolution or 331.5±10 mT for CYPMPO-containing solution; modulation ofmagnetic field, 0.079 mT; time constant, 0.10 sec; sweep time, oneminute for DMPO-containing solution or four minutes forCYPMPO-containing solution; output power, 8.0 mW. The obtained ESRspectrum derives from generation of DMPO spin adduct or CYPMPO spinadduct trapping active oxygen and free radical species in a sample. Theconcentrations of active oxygen and free radical species in the samplewas determined based on signal intensities in the ESR spectrum of thespin adduct. Since a transition metal, Mn gives an ESR spectrum usefulas a reference ESR signal, it is possible to precisely determine an ESRsignal intensity of DM PO spin adduct (or CYPMPO spin adduct) trappingactive oxygen and free radical species, relative to that of Mn, forprecise determination of the concentrations of active oxygen and freeradical species. The ESR analysis is specifically described below.

In preparation of ESR sample solution, 100 μM ferrous sulfate, 100 μMdiethylenetriaminepenta-acetic acid (DETAPAC), 5 mM CYPMPO, thecomposition of Example 1 or Trolox with various concentrations, and 100μM hydrogen peroxide were added into 200 mL phosphoric acid buffer (pH:7.8) which was significantly supplied with nitrogen gas. In preparationof the sample, the above substances except hydrogen peroxide were mixedwith each other in advance, and then hydrogen peroxide was supplied tothe resultant solution, so as to generate hydroxyl radical throughFenton reaction between hydrogen peroxide and iron ion. ESR measurementwas initiated by sweeping magnetic field one-minute after hydrogenperoxide was added, so as to keep constant the reaction time forgenerating a predetermined amount of hydroxyl radical. The generatedhydroxyl radical was trapped by CYPMPO in the ESR sample solution toform the spin adduct (CYPMPO-OH) which gives an eight-line ESR spectrum.The hydroxyl radical concentration in the ESR sample solution wasdetermined in terms of a relative intensity of CYPMPO-OH/Mn ESR signal,by comparison in ESR spectral intensity between a peak in fourth lowestmagnetic field in the CYPMPO-OH ESR spectrum and a peak in second lowestmagnetic field in six-line Mn ESR spectrum. The relative intensity ofCYPMPO-OH/Mn ESR signal for the sample solution containing thecomposition of Example 1 or Trolox was determined, with respect to thatfor a control solution not containing the composition of Example 1 orTrolox. The hydroxyl radical-scavenging activity of the oral dosagecomposition in Example 1 was determined in terms of variation in therelative intensity of CYPMPO-OH/Mn ESR signal obtained for the ESRsample solution containing the composition of Example 1.

The hydroxyl radical-scavenging activity of this composition of Example1 was determined to be 960 μg/ml in terms of the concentration of thiscomposition of example 1 (the final concentration in the ESR samplesolution) causing 50% reduction of the relative intensity ofCYPMPO-OH/Mn ESR signal compared to that for the control solution. Thedetermined hydroxyl radical-scavenging activity corresponds to 0.43 μmolTrolox equivalent /mg in terms of the chemical equivalent of Trolox.Accordingly, this composition of Example 1 proved to have a sufficientactivity with high health functionality.

<Example of Foods Each Containing the Dried Spirulina Powder of the OralDosage Composition in the Present Invention (Examples 2 and 3)>

The dried spirulina powder was mixed with a separate-type dressing, forpreparation of the oral dosage composition in Example 2. The driedspirulina powder is a spirulina powder available from Spirulina Bio-LabCo., Ltd. The separate-type dressing is a commercialized product ofKewpie Corporation. The resultant products contain the dried spirulinapowder with different contents of 0.01%, 0.1%, 1.0% and 5.0% by weightbased on that of the separate-type dressing.

For preparation of the oral dosage composition in Example 3, the samedried spirulina powder as in the Example 2 was mixed with a noodlecommercially available. The resultant products contain the driedspirulina powder with different contents of 0.01%, 0.1%, 1.0% and 5.0%by weight based on that of the noodle.

The products of Examples 2 and 3 were evaluated by ten evaluators. Allof the dressings and separate-type dressings containing dried spirulinawere judged to be edible by all evaluators, thereby the presentinvention was confirmed to have a superior quality in respect offunctional evaluation.

However, this evaluation revealed that the 0.01 wt % product needs to beconsumed in high amount for giving sufficient effectiveness againstNASH, thereby showing the 0.01 wt % product is not suitable forpractical use in respect of functional evaluation. This evaluationrevealed that the oral dosage composition needs to contain the driedspirulina with a content of 0.1 wt or more.

<The Effectiveness of the Oral Dosage Composition of the PresentInvention Against NASH>

The oral dosage compositions of the present invention were administeredto NASH biological and pathological model rats, which are raised fromfatty liver rats each loaded with oxidative stress (OS) under biologicalhypoxic condition, in order to evaluate the effectiveness of the oraldosage composition of the present invention against NASH. The evaluationwas performed in terms of biochemical variation in blood of the rats,ROS-derivative variation of the liver mitochondria, and pathologicalvariation in liver tissue.

NASH pathological model was prepared from 6-week-old Wister male ratseach having a weight of 180 g to 200 g, in accordance with Takayama'smethod (see non-patent reference 1). For preparation of fatty liverrats, a choline-deficient food (CDHF, available from Oriental Yeast Co.,Ltd.) was administered to the male rats in free-feeding for four weeks.CDHF was administered to the rats continuously after four weeks, formaintaining pathological model.

The animals were raised in a polypropyrene opaque cage (width 220,length 320, height 135, available from Natsume Seisakusho Co. Ltd) under40 to 50% humidity at a temperature of 20° C. to 25° C., with acontrolled 12/12 hour light-dark cycle (light on AM 8:00, light off PM8:00).

For preparation of NASH pathological model rats each loaded withoxidative stress (OS) under biological hypoxic condition, sodium nitritein physiological salt solution was intraperitoneally administered to thefatty liver rats at 30 mg/kg (body weight) per day for six weeks. Duringthis administration, blood test in tail vein was performed in two-weekintervals, for confirmation of normal development of pathologicalcondition.

The oral dosage composition in Example 1 of the present invention wasorally administered to the fatty liver rats at 2 g/kg (body weight) perday or 6 g/kg (body weight) per day for six weeks, in free-feeding,instead of the sodium nitrite solution, for evaluation of effectivenessof the oral dosage composition in the present invention against NASH.

After the administration, the rats were sacrificed for observation ofbiochemical variation in blood thereof, variation in amounts of livermitochondria-produced active oxygen and free radical species (ROS), andpathological variation in liver tissue.

Explanations are give as to this Example, with reference to group 1(CDHF+OS), group 2 (CDHF+OS+2 g/kg of Example 1), group 3 (CDHF+OS+6g/kg of Example 1). Eight rats were used for each group (n=8).

AST and ALT values in plasma was determined with transaminase CII—testWako (available from Wako Pure Chemical Industries).

The liver mitochondria-produced active oxygen and free radical species(ROS) were detected in the following procedure. The liver was perfusedwith 1.15% potassium chloride solution (containing 5 mM benzamidine)through inferior vena cava, and then sampled. The sampled liver tissue 1g was added to a tris hydrochloric acid buffer solution (pH 7.4,containing 0.25 M sucrose and 0.1 M potassium chloride), and thenhomogenized. Next, the resultant solution was subjected to centrifugalseparation at 3000×g for 10 minutes at 4° C. to obtain a supernatantliquid. Subsequently, the obtained supernatant liquid was subjected tocentrifugal separation at 9000×g for 20 minutes at 4° C., so as toobtain a precipitation product. The precipitation product wascentrifugally washed with tris hydrochloric acid buffer solution (pH7.4, containing 0.25 M sucrose and 0.1 M potassium chloride) two times,giving mitochondria fractions. Each mitochondria fraction 14.28 mg wasdiluted with 1 ml of tris hydrochloric acid buffer solution (pH 7.4,containing 0.25 M sucrose and 0.1 M potassium chloride), so as to obtaina sample solution having a concentration of 500 μg/ml in terms ofmitochondria protein.

For determination of amounts of produced active oxygen and free radicalspecies (ROS), the obtained liver mitochondria solution 35 μl was mixedwith 25 μl of a solution (containing 0.1% dodecyl maltoside, 5 mMglutamate, 5 mM malate, and 200 mM succinate), 20 μl of 4.6 M DMPOsolution and 20 μl of 2 mM NADH solution 20 μl, and then incubated at37° C. for five minutes. The solution was measured with an ESR apparatus(JESREIX/HR available from JEOL Ltd.), soon after incubated.

Prior to the ESR measurement for the solution, ESR spectral intensity ofDMPO spin adduct (DMPO-OH) was determined relative to that of Mn²⁺ inMnO disposed inside the cavity. Namely, ESR spectral intensity of DMPOspin adduct was determined in terms of relative intensity with respectto that of Mn²⁺ (DMPO-OH/Mn).

The ESR spectral data was statistically analyzed by one-dimensionalanalysis of variance (ANOVA) and subsequent Turkey's multiple-comparisontest to be evaluated in terms of “mean value±standard error”. Thecomparison between two groups was analyzed by student's t-test,significance level of 5% or less was determined to be significant.

After the administration, the rats were sacrificed, for observation ofbiochemical variation in blood of the rats, ROS-productive variation atthe liver mitochondria and pathological variation in liver tissue so asto give the following results.

Biochemical measurement tests were performed for group 1 notadministered with the dried spirulina product (CDHF+OS), for obtainingthe following results regarding variations in biochemical propertiesbefore and after administration.

AST value (units/ml) in plasma was found to rise to 140±7 IU/L from 23±2IU/L.

ALT value (units/ml) in plasma was found to rise to 16±2 IU/L from 7±2IU/L.

The above results show NASH symptom due to oxidative stress appears.

Biochemical measurement tests were performed for group 2 administeredwith 2 g/Kg (body weight) (CDHF+OS+2 g/Kg of Example 1), for obtainingthe following results regarding variations in biochemical propertiesbefore and after administration of 2 g/Kg (body weight) of Example 1.

AST value (units/ml) in plasma was found to rise to 100±20 IU/L from25±2 IU/L.

ALT value (units/ml) in plasma was found to rise to 14±3 IU/L from 8±2IU/L.

The above results demonstrate that NASH symptom due to oxidative stressis alleviated by administration of 2 g/Kg (body weight) of the oraldosage composition in Example 1 of the present invention.

Biochemical measurement tests are conducted for group 3 administeredwith 6 g/Kg (body weight) (CDHF+OS+6 g/Kg of Example 1), for obtainingthe following results regarding variations in biochemical propertiesbefore and after administration of 6 g/Kg (body weight) of Example 1.

AST value (units/ml) in plasma was found to rise to 35±5 IU/L from 21±3IU/L.

ALT value (units/ml) in plasma was found to rise to 10±2 IU/L from 7±3IU/L.

The above results demonstrate that NASH symptom due to oxidative stressis alleviated by administration of 6 g/Kg (body weight) of the oraldosage composition in Example 1 of the present invention.

Hematoxylin-eosin stain was employed to color macrovesicular steatosisin conventional way for confirmation of the pathological variation inliver tissue, giving the following results regarding the accumulation ofmacrovesicular steatosis.

Group 1 (CDHF+OS) proved to suffer from advanced macrovesicularsteatosis and substantial disarray of liver cells.

Group 2 (CDHF+OS+2 g/Kg of Example 1) proved to suffer from moderatemacrovesicular steatosis and moderate disarray of liver cells.

Group 3 (CDHF+OS+6 g/Kg of Example 1) proved to suffer fromless-advanced macrovesicular steatosis and slight disarray of livercells.

The above results demonstrate that the macrovesicular steatosis in livertissue and disarray of liver cells developing with NASH are alleviatedby administration of the composition in Example 1, depending on itsconcentration.

Masson trichrome stain was employed to color collagen fibril inconventional way for confirmation of the pathological variation in livertissue, giving the following results regarding collagen fibril.

Group 1 (CDHF+OS) proved to suffer from bridging between portal regionand central vein region, and pseudolobule formation (F3˜F4).

Group 2 (CDHF+OS+2 g/kg of Example 1) proved to suffer from slightbridging between portal region and central vein region.

Group 3 (CDHF+OS+6 g/kg of Example 1) proved to suffer from fibrosis inportal region and in the vicinity of central vein, without bridging.

The above results demonstrate that the bridging and fibrosis developingwith NASH are alleviated by administration of the composition in Example1, depending on its concentration.

Berlin blue stain was employed to detect iron ions in conventional wayfor confirmation of the pathological variation in liver tissue, givingthe following results regarding coloring due to iron precipitation.

Group 1 (CDHF+OS) proved to suffer from substantial iron precipitationbetween portal region and central vein region.

Group 2 (CDHF+OS+2 g/kg of Example 1) proved to suffer from slight ironprecipitation between portal region and central vein region.

Group 3 (CDHF+OS+6 g/kg of Example 1) proved to hardly suffer from ironprecipitation.

The above results demonstrate that the iron precipitation in livertissue developing with NASH are alleviated by administration of thecomposition in the present invention, depending on its concentration.

The above results using NASH model animals confirm the dried spirulinaproducts efficacy for anti-oxidative treatment against NASH, one oftypical life style-related diseases.

<Suppression Effects of Liver Mitochondria-Produced Free Radicals withthe Use of the Oral Dosage Composition in the Present Invention>The following tests were performed for confirmation of effectiveness ofthe oral dosage composition in the present invention, in terms ofsuppression of active oxygen and free radical generation resulting fromenergy metabolism in the liver mitochondria of NASH pathological animalmodel.

The following experiments were performed for rats in groups 1, 2 and 3.NASH pathological model was prepared in accordance with the aboveTakayama's method (see non-patent reference 1).

Mitochondria fractions were obtained from rats in groups 1, 2 and 3.(See Egashira T. et al: Toxicology Letter, 117, 115-119 (2000)) That is,the liver sample was obtained by perfusing the liver with 1.15%potassium chloride solution (containing 5 mM benzamizine) through aninferior vena cava. The liver tissue 1 g was added to a trishydrochloric acid buffer solution (pH 7.4, containing 0.25 M sucrose and0.1 M potassium chloride), and then homogenized. Next, the resultantsolution was subjected to centrifugal separation at 3000×g for 10minutes at 4° C. to obtain a supernatant liquid. Subsequently, theobtained supernatant liquid was subjected to centrifugal separation at9000×g for 20 minutes at 4° C., so as to obtain a precipitation product.The precipitation product was centrifugally washed with trishydrochloric acid buffer solution (pH 7.4, containing 0.25 M sucrose and0.1 M potassium chloride) two times, giving mitochondria fractions.

Subsequently, mitochondria produced-reactive oxygen and free radicalspecies in each group were trapped by a spin-trapping agent, DMPO, toobtain DMPO spin adduct in the following way (see, Yudong Wang et al:Free Radical Biology and Medicine, 36(11), 1434-1443 (2004)). The DMPOspin adduct was detected by ESR spectroscopy. DMPO, mitochondria andNADH were ice-cooled in advance. Other substances were warmed at 37° C.in advance. The assay solution was prepared by adding 35 μl ofmitochondria emulsion (containing 0.5 mg of protein), 920 mM of DMPO,0.1% dodecylmaltoside, 10 mM of potassium L-glutaminate, 10 mM ofL(−)-sodium malate, 200 mM of disodium succinate and 100 μM of NADH to abuffer solution (pH 7.4) containing 5 mM of trisaminomethane, 0.25 mM ofsucrose and 0.1 mM of potassium chloride. Then, the resultant solutionwas incubated at 37° C. for 5 minutes, for undergoing ESR measurement.

The mitochondria fraction in each group was suspended with 0.03 Mtris-hydrochloric acid buffer solution (pH 7.4, containing 0.25 Msucrose and 0.1 M potassium chloride), and then immediately preserved at−80° C. prior to undergoing ESR analysis.

The composition in Example 1 was added to the ESR measurement samplesolution for ESR spectroscopic analysis. The composition in Example 1was examined for evaluation of its efficacy of reducing active oxygenand free radicals (ROS) which are produced due to a disorder ofmitochondrial function, in terms of variation in an intensity of ESRspectrum of DMPO spin adduct trapping active oxygen and free radical(see Takayama F. et al: Japanese Journal of Pharmacology, 85, 227-233(2001)). Specifically, the presence of mitochondria produced-activeoxygen and free radicals (ROS) was confirmed by observation ofvariations in intensities of ESR spectra of DMPO and DMPO-OH trappingactive oxygen and free radicals (ROS) with their typical g-values andfine structure parameters. The dried spirulina product was added to theassay solution containing NASH pathological mitochondria. The obtainedsolution was examined using ESR spectroscopy in terms of variation inrelative intensity of ESR spectra (DMPO-OH/Mn), for evaluation of itsefficacy of reducing active oxygen and free radical species which aregenerated due to energy metabolism of NASH-pathological model. Theefficacy was evaluated in terms of IC₅₀ (μg/ml) representing theconcentration of this composition of example 1 causing 50% reduction ofthe relative ESR signal intensity. The ESR apparatus was operated forESR measurement in the same way as in “the effectiveness of the oraldosage composition of the present invention against NASH” describedabove.

The following results were obtained as to the relative signal intensityof ESR spectra arising from the presence of liver mitochondria-producedROS. The relative signal intensity was determined to be 1.0±0.1 in group1 (CDHF+OS), 0.8±0.1 in group 2 (CDHF+OS+2 g/Kg of Example 1), and0.6±0.1 in group 3 (CDHF+OS+6 g/Kg of Example 1), increasing from0.5±0.1 obtained prior to an addition of oxidative stress. The resultshows that the amount of liver mitochondria produced-ROS increasing withNASH development is reduced by administration of the composition inExample 1, depending on its concentration.

The above results demonstrate that the oral dosage composition in thepresent invention enables to suppress and regulate the radicalgeneration resulting from energy metabolism of liver mitochondria ofNASH pathological animal model. Therefore, the oral dosage compositionin the present invention proved effective in preventing, alleviating,and curing NASH when orally administered to patients.

1. An oral dosage composition for prevention or treatment ofnon-alcoholic steatohepatitis, said oral dosage composition comprisingspirulina as an effective ingredient, wherein said oral dosagecomposition has a hydroxyl radical scavenging activity with IC₅₀ valueranging from 3000 μg/ml to 100 μg/ml determined by electron spinresonance spectroscopy—spin trapping method.
 2. (canceled)
 3. The oraldosage composition as set forth in claim 1, comprising 0.1% or more byweight of a dried spirulina.
 4. (canceled)